Insect cells are widely utilized for the production of heterologous glycoproteins, which are proteins that include oligosaccharide attachments. Many of the most valuable therapeutics are glycoproteins and the composition of the attached oligosaccharide can significantly impact the properties and value of these therapeutics. In particular, the presence or absence of sialic acid on an oligosaccharide can alter the glycoprotein's structure, stability, biological activity, and in vivo circulatory half-life. Glycoproteins containing oligosaccharides missing sialic acid are removed from human circulation and this rapid clearance will diminish the therapeutic effectiveness of a glycoprotein biopharmaceutical. Unfortunately, insect cells do not generate significant levels, if any, of sialylated glycoproteins, and this inability to sialylate severely limits the further application of the insect cell expression system. The objective of this project is to manipulate the metabolic pathways in insect cells so that these cells will produce high levels of complex, fully sialylated glycoproteins. A critical sialylation pathway involves the post-translational addition of the donor sialic acid substrate, cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac or CMP-sialic acid) onto a specific acceptor carbohydrate substrate ending in galactose (Gal) via a reaction catalyzed by the enzyme sialyltransferase. Each of these three critical components (donor substrate, acceptor substrate, and sialyltransferase enzyme) is limiting or absent in insect cell lines so metabolic engineering strategies will be implemented to eliminate the bottlenecks. Limitations in CMP-Neu5Ac will be overcome by expressing essential enzymes in order to complete the intracellular synthesis of the donor substrate. Expression of heterologous glycosyltransferase enzymes, suppression of unfavorable processing reactions, and examination of alternative insect cell hosts will overcome limitations in Gal acceptors. Combining these pathway modifications with sialyltransferase expression should ensure full sialylation in insect cells. Completion of this project will lead to an increase in the number of expression systems that can produce the high-value sialylated therapeutic glycoproteins desired by the health care community. In this way, sialylation pathway engineering may increase glycoprotein quality and lower health care costs for patients receiving these therapeutics.